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Studies on Ulcerative Disease Caused by
ProvidenciastuartiiBacteria in Indian Major
Carp, Labeo rohita (Ham.)

Abstract

Rohu (Labeo rohita) is a species of fish of the carp family cyprinidae. A survey was carried out from September, 2012 to August, 2013 to study the prevalence of bacterial ulcerative disease in L. rohita, cultivated in a freshwater farm located at Moongilthuraipattu Village of Villupuram District, Tamil Nadu, India. To mimic the infection in healthy fish, organs such as liver, gill and ulcerative skin were used to prepare the inoculum in PBS buffer. The inoculum was injected into healthy animals through intramuscular injection, immersion challenge and oral route. The tissue supernatant from infected rohu fish was screened for isolation and screening of the causative organism responsible for ulcerative lesion in L. rohita. Based on the biochemical, morphological and molecular features the bacterial culture isolated from infected rohu was tentatively identified as Providencia species like bacterium which was further confirmed by the analysis of its 16S rRNA gene using PCR and DNA sequencer and it has been identified as P. stuartii. Further, its partial sequence was deposited in GenBank (KF155520.1).

Keywords

INTRODUCTION

Providencia species are found in multiple animal reservoirs, including flies, birds, cats, dogs, cattle, sheep, guinea
pigs, penguins, and are resident oral flora in reptiles such as pythons, vipers, and boas. Providencia species are also
found commonly in soil, water and sewage. Examples of Providencia infections in animals include neonatal diarrhea
due to P. stuartii infection in dairy cows and enteritis caused by P. alcalifaciens infection in dogs. P. rettgeri has been
isolated in crocodiles with meningitis/septicemia and in chickens with enteritis [1]. P. heimbachae has been isolated in
penguin feces and from an aborted bovine fetus [2]. The genus Providencia, belonging to the family
Enterobacteriaceae, consists of 9 species, namely P. alcalifaciens, P. stuartii, P. rettgeri, P. rustigianii, P. heimbachae,
P. vermicola, P. sneebia, P. burhodogranarieaand P. thailandensis [1], [3-6]. In human, Providencia species have been
isolated from urine, stool, blood, sputum, skin and wound cultures. One case study has described P. stuartii as the
etiology of infective endocarditis,[7].Papadogiannakis [8] studied on the P. stuartii infection with severe skin
ulceration and cellulitis isolated from a dog.Among Providencia species, P. rettgeri is the only species isolated from
farmed fish (Hypophthalamichthysmolitrix) in Israel. Till date, it is the only report implicating this organism as a fish
pathogen[9]. In view of acknowledging this, P. vermicola was the first species isolated from freshwater fish, L. rohita
and its partial 16S rRNA sequence was deposited in GenBank by the authors of this study (accession no. KF155518.1.).
In the present study, moribund young L. rohita with clinical signs of ulcer on the abdomen and pectoral fin surfaces
was observed in freshwater fish farms. A work was undertaken to isolate the causative organism responsible for
mortality of Indian major carp, rohu. Experimental infection for high mortality of L. rohita was carried out to reproduce
the infection in healthy fish using the bacterial cultures isolated from the infected fish. Llobrera and Gacutan[3] studied
the Aeromonas hydrophila associated with ulcerative disease epizootic in Laguna de Bay, Philippines. John
Thomas[11] studied the ulcerative disease caused by A. caviae-like bacterium in Indian catfish, Clariasbatrachus. The gram negative, rod shaped-bacterium isolated from Indian freshwater carp, L. rohita was tentatively identified as P.
stuartii. In order to acquire more information on the taxonomic position, DNA from the Providencia sp. was isolated,
polymerase chain reaction was performed and 16S rRNA gene of the sample was amplified and sequenced to confirm
the species affecting the freshwater fish, L.rohita. The partial gene sequence of 16S rRNA belongs to P. stuartii was
deposited in GenBank, with accession no. KF155520.1

MATERIALS AND METHODS

A. Collection and Maintenance of Naturally Infected L. rohita

The moribund freshwater fish L. rohita (56-69g) with the signs of red ulcers on the abdomen and base of
pectoral fin were collected from a fish farm located at Moongilthuraipattu Village of Villupuram District, Tamil Nadu
and transported within three hours at 30°C in live state to the laboratory in an aerated polythene bag. In laboratory, the
infected animals were maintained in 75 L aquarium tanks with tap water at a temperature of 27-30°C. The animals were
fed with commercial fish feed.

B. Physico-chemical Parameters of Water

The physico-chemical parameters of the pond water such as temperature, pH, dissolved oxygen (DO),
biochemical oxygen demand (BOD) and chemical oxygen demand (COD) of the pond were determined following
standard protocols [12].

The organs liver, gill and ulcerative skin from naturally infected fish were dissected out and homogenized with
sterile PBS buffer. The diluted samples were plated on nutrient agar, Aeromonas agar and trypticase soy agar by spread
plate technique and incubated at room temperature for 24-48 h. Overriding colonies (from trypticase soy agar) were
selected and again streaked on trypticase soy agar for locating pure cultures. These pure cultures were maintained on
trypticase soy agar for further biochemical and molecular characterization studies. Biochemical characterization was
done and the bacterial isolates were identified according to Bergey’s manual of Determinative Bacteriology[13].

D. Molecular Identification of Bacterial Strain

1) Bacterial Genomic DNA Isolation:

Genomic DNA was extracted and purified by following the standard techniques [14], [15] with slight
alterations. Luria broth was inoculated with a loop full of pure culture and incubated overnight. From this, 1.5 mL of
grown culture was centrifuged at 10000 xg for 30 minutes, the resulted pellet was suspended in 600 μL of TE buffer.
To this, 45 μL of 10% SDS was added followed by addition of 5 μL lysozyme and mixed well. The mixture was
incubated for 1 h at 37° C. After incubation, 500 μL of phenol: chloroform was added and the mixture was mixed well
by inverting the tube until the formed two phases completely mix with each other. Then the sample was centrifuged at
10000 xg for 15 min. The aqueous phase was transferred to a new centrifuge tube, to this equal volume of phenol:
chloroform was again added and centrifuged at 10000 xg for 15 min. The resulted aqueous phase was mixed with 50
μL of 3M sodium acetate in a new micro centrifuge tube and mixed well. To this, 300 μL of isopropanol was added and
mixed gently to precipitate the DNA, then the mixture was centrifuged at 10000 xg for 10min. The resulted pellet was
washed with 70% ethanol for 30 sec and centrifuged at 8000 xg for 1-2 min. The obtained pellet was resuspended in
100 μL of TE buffer and stored at 4° C for further analysis. Then the sample (5 μL) was allowed to run in 0.8% agarose
gel to confirm the presence of bacterial genomic DNA under UV light.

Disease-free, Labeo rohita were collected from the Department of Fisheries, Mettur Dam, Tamil Nadu with no
record of ulcerative syndrome. The live fish were transported to laboratory in an aerated bag and maintained in 700 L
FRB tank with continuous aeration at room temperature (27- 30°C) with tap water. The animals were fed twice a day
with commercial fish feed.

F. Reproduction of Bacterial Infection

Ulcerative skin from the naturally infected fish was cut and homogenized with PBS buffer. The homogenized
sample was centrifuged at 1000 xg for 10 min at 4° C. Healthy and active fish were injected intramuscularly with 5U of
suspension and maintained for a period of 7days. The control fish were injected intramuscularly with 5U of sterile PBS.
Experimental fish were examined often for clinical signs of disease and mortality.

G. Experimental Infection of Bacterial Isolate in Healthy L. rohita

Infectivity of the new bacterial isolate from the naturally infected fish was studied in healthy individuals.
Immersion, intramuscular and oral route of administrations were followed to determine the mode of infection and the
pathogenicity of bacterial isolate in healthy carp, Labeo rohita based on the standard protocols [16], [17].

H. Preparation of Bacterial Inoculum

The bacterial cultures isolated from naturally infected fish was grown on nutrient agar for use in pathogenicity
experiments. The pathogenicity of the bacterial isolate was tested by bath immersion, intramuscular injection and oral
administration. The bacterial count was determined by standard dilution and plating methods [18].

I. Experimental Infection by Immersion Method

The experiment was carried out by following [11] with some modifications. Healthy fish (12 fish per tank)
were reared in aquarium tanks of 75 L capacity containing sterilized freshwater with continuous aeration. Air stones
and air tubes were sterilized by immersing them in 5% sodium hypochlorite and by washing them thoroughly with
sterilized water before use. The tanks were covered to prevent contamination. Aseptic techniques were used throughout
the experiment. Fish were fed with commercial fish feed. For the experimentally induced infection, the fish were
exposed to different concentrations of bacterial cells (103,104, 105, 106 and 107 CFU ml
−1). The control consisted of fish
exposed to sterilize freshwater alone.

J. Infection by Intramuscular Injection

The experiment was carried out by following [11] with some modifications. Fish (12 per dosage and tank)
were maintained in 100 L FRB tank containing sterilized freshwater at room temperature. The bacterial isolate was
inoculated into healthy fish through intramuscular injection, near the dorsal fin at doses of 103,104, 105, 106 or 107 CFU
per animal. Control fish were inoculated only with sterile PBS buffer.

K. Oral Infection

Fish were individually secluded in the aquarium tanks and starved for 24 h. Each fish was fed with a piece of
fish meat which was injected with 1 ml of bacterial suspension (1012 CFU). The fish were fed thrice, with an interval of
8 h. After the last feeding with infected meat, the animals were fed with non-infected meat for 7 days. In the control
group, fish were fed only with non-infected meat. In all experiments, animals were examined once in 8 h a day for
clinical signs of disease and mortality.

L. Confirmation of pathogenicity

The specific action of the bacterial sample isolated from naturally infected fish as a pathogen was confirmed
by re-isolating the bacterium from the liver, gill and ulcerative skin of moribund fish to fulfil Koch's postulates. The
samples were inoculated on trypticase soy agar plates by spread plate technique for the isolation of bacterial pathogen.
The isolated bacteria were recognized using the earlier described procedure.

RESULTSANDDISCUSSION

The physico-chemical features of the fish farm water were determined. The ranges of temperature, pH, DO, BOD
and COD were: 26-30 °C, 6.3-8.1, 5.7-7.1 mg/L, 2.36-2.89 mg/L and 183-209 mg/L, respectively. A high temperature
of 30 °C was recorded during summer. Death of fish due to bacterial pathogen extended to 99-100% within 2-3 days
after the presence of ulcer on the surface of infected fish samples. The clinical signs of the diseased fish contained
ulcerative lesions on the abdomen/body surface and bases of the pectoral fin with reddish color on the surface of the
infected portions.The bacterial inoculum prepared from ulcerative tissue samples of diseased fish alone produced
clinical indication of ulcerative abrasion in the disease free, healthy L. rohita fish under experimental condition. Studies
were carried out on different organs like ulcerative skin, liver and gill acquired from the infected fish samples. Single
prevailing bacterial culture isolated from each of the media was tested for their infectivity in the healthy L. rohita, but
the bacterial isolate from trypticase soy agar and nutrient agar alone resulted mortality and reproduced the signs of
ulcerative lesions in healthy L. rohita. This specific bacterial isolate alone was selected and identified based on the
colony morphology, biochemical, physiological and molecular identification. Colonies grown on TSA plates were
Gram-negative rods, motile, circular, 2.1 – 2.2 mm in diameter, slimy and convex. Colonies are smooth with entire
edges and an intense characteristic smell was produced with growth on trypticase soy agar. The biochemical
characterization of the bacterial culture resulted in positive for citrate, inositol, glycerol, gelatin, sorbitol, lyxose, DMannose,
D-Serine, catalase, indole, phenyl alanine, glucose, nitrate reduction, trehalose, and methyl red. Negative
results for D-Mannitol, D-Xylose, cellobiose, esculin, sorbitol, pigmentation, oxidase, lactose, arginine dihydrolase,
lysine decarboxylase, ornithine decarboxylase, H2S, Voges-Proskauer, gas from glucose, L-Arabinose, maltose,
raffinose, salicin, D-Xylose, deoxyrinonuclease and lipase.Based on thebiology, biochemical characterization and
morphology of the isolated bacteria from infected L. rohita was tentatively identified as P. stuartii like bacterium.
Further confirmation of P. stuartii– like bacterium by analyzing its 16S rRNA gene was performed using PCR. For
this, bacterial universal primers 27f & 1525r were used for the amplification of 16S rRNA gene and the results
wereshown in Fig.1. The sequencing of the strain discovered a homology of 99% with P. stuartii. Hence it is a
Providencia sp. with features of P. stuartii.

The exposure of L. rohita to Providencia like bacterium was tested by immersion, intramuscular injection and oral
infection. The maximum concentration of Providencia sp. like bacterium (30 x 106 CFU ml-1) caused 33.33, 43.33,
56.6, 63.3 and 87.3% of mortalities through immersion method at 18, 30, 48, 84 and 108 h of post exposure,
respectively. The LC50 value of Providencia sp. like bacterium was determined. It was found to be 3.22 x 105, 3.69 x
105 and 5.85 x 106 CFU ml-1 at 18, 36 and 54 h of post injectionrespectively.The highest concentration, 54 x 105 and 54
x 106 possible cells of Providencia like bacterium per animal caused 100% death within 96 and 60 h of post infection,
respectively. During bath exposure, whereas the lower presentation of 54 x 103 and 54 x 104 feasible, study bacterial
cells, per animal caused 43.33 and 73.3% death within 120 and 108 h of post infection. The LD50 value of Providencia
sp. like bacterium for intramuscular course was defined at different time intervals and was found to be 2.1 x 106 and
1.04 x 107 per animal after 72 and 96 h of post injection, respectively. There was no mortality in the oral route
administered L. rohita fish. The infection of Providencia sp. like bacterium was established by satisfying Koch’s
postulate.

Rohu is considered to be the high number of fish cultivated than other major carps in India. It has high market
value and are easily available for culture. The risk of diseases in rohu also increased in making the rohu susceptible to
diseases in association to the exotic carps [18]. Diseases are the most serious limiting factors in aquaculture, because of
increased density of fish in restricted water where the fish pathogens can transmit from one to another [19]. The rohu is being mainly affected by bacterial pathogens. Among bacterial pathogen, ulcer disease is the major one, followed by
columnaris and dropsy.Among bacterial pathogens, P. rettgeri was one of them which caused a mass mortality among
farmed silver carp, Hypophthalmichthysmolitrix in Israel during 1976. Till date, this has been the only report
associating this organism as a fish pathogen [9]. This disease tempted severe epidemics of ulcerative diseases in farmed
fish. From 2012, an ulcerative disease which is caused by a bacterium has been witnessed in nursery and grow-out
ponds located in the Villupuram District of Tamil Nadu, India.The clinical signs of infected rohu fish include large red
ulcer lesions on the abdomen and base of pectoral fin. The indications in the infected rohu fish agrees with the reports
of [20], [11] who have reported a related clinical indication in other bacterial infections. Isolation and biochemical
characterization were performed on organs such as liver, gill and ulcerative skin from infected rohu using nutrient agar
(NA), Aeromonas agar and trypticase soy agar (TSA). Single pure colony was isolated from TSA and NA plates which
exactly revealed the presence of culture colony in the specific medium, which was again studied in healthy rohu fish to
confirm that the bacterial sample from infected rohu fish was a pathogen in order to fulfil the Koch’s postulates. Many
authors have reported that, Aeromonas and Pseudomonas spp. are causing major infections in L. rohita (FAO, 2007)
which persuade with severe lesions of ulcerative diseases in fish from Southeast Asia especially in India.In afford with
colony morphology, biochemical and molecular identifications the disease caused in L. rohita was identified as
Providencia sp. like bacterium. This is the second report in L. rohita for the presence of Providencia sp. occurring
naturally in cultured freshwater fish. Hence, only one report was available about the infection of P. rettgeri isolated and
studied in silver carp, H. molitrix in 1976 reported by [9]. Similar ulcerative lesion induced by Aeromonas sp. in many
fishes were stated in many reports [21], [22], [11]. Hence in the present study, the bacterial culture Providencia sp. like
bacterium was confirmed as a pathogen to satisfy Koch’s postulates in normal rohu fish. DNA isolation and PCR were
executed to detect the bacterial strain at molecular level using universal primers specific to 16S rRNA gene of
prokaryotes. The application of 16S rRNA gene sequence is to study the bacterial phylogeny and taxonomy which
include important reasons like (i) its presence in almost all bacteria, often existing as a multigene family, or operons;
(ii) the roles of the 16S rRNA gene over time has not changed, recommending that random sequence changes are a
more precise degree of time (evolution); and (iii) the 16S rRNA gene (1500bp) is large enough for informatics purposes
[23], [11]. The PCR band (~1500bp) confirmed the presence of Providencia sp. Species identification was done using
16S rRNA gene sequencing. Unfortunately, on no account definition for species identification using 16S rRNA gene
sequencing exhibited. In not any studies does the definition of a species “match” ever exceed 99% similarity [11]. The
species of Providencia was identical as P. stuartii and it (Providencia) is the second report for its infection in L. rohita,
its 16S rRNA gene partial sequence was deposited in GenBank with accession no. KF155520.1. Whereas, the
bacterium P. vermicola was first isolated and reported from India by [4] from an infected juvenile nematode,
Steinernemathermophilum. The authors of this study have submitted the 16S rRNA partial sequence of P. vermicola
bacteria from L. rohitain GenBank with accession no. KF155518.1. The responsibility of rohu fish, L. rohita to P.
stuartii was tested by bath exposure (immersion), intramuscular injection and oral route in L. rohita. Finally, the
significant gateway of the pathogen is by means of penetration of tissue at the site of wounds or injuries. The expiry by
the above experiment showed that the pathogenicity of P. stuartii in L. rohita depends mainly on the doses and period
of exposure.

CONCLUSION

Bacterial diseases are responsible for high mortality in both wild and cultured farm fish. The real role of
microorganisms vary from a major pathogen to that of an opportunistic pathogens which makes its host organisms
(fish) moribund by commencing infection development. Bacterial flora of fish is directly proportional to its
environment. In the present study, clear variations in physico-chemical parameters has been observed which may be
considered as conducive condition for the outbreak of disease. Biochemical and molecular characterization of the
species confirmed that it is the first report, species stuartii of genus Providencia causing infection and mortality in the
freshwater fish,L. rohita infection. From the pathogenicity experiments it is evident that the main portal entry of the
pathogen is by means of penetration of tissue at the site of rashes or wounds and the mortality of experimentally
infected animals depends on the dosage and time period of exposure.

ACKNOWLEDGEMENT

One of the authors[1] thank the Department of Science & Technology, New Delhi, India for providing
INSPIRE Fellowship to carry out this study. And the authors of this research work are thankful to the authorities of
host Institute for providing necessary facilities throughout the work.